Xylanase, endo-1,4-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2010 to January 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 and 100 mg TOS/L (corresponding to 22.2 mg aep/L)
- Sampling method: At the start of the definitive test, three samples (10 mL) were taken from the freshly-prepared control and test media. After 72 hours, the contents of the replicate flasks for each group were pooled and further samples taken for analysis. Additional samples were also taken from a flask containing Xylanase, PPQ 30189 at a nominal concentration of 100 mg TOS/L but with no algal cells, in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells.
- Sample storage conditions before analysis: Frozen

Vehicle:

no

Details on test solutions:

Method: The test substance (957.4 mg) was added directly to algal culture medium (1000 mL) to provide the test medium at a nominal concentration of 100 mg TOS/L.
- Controls: Culture medium.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae & Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland
- Method of cultivation:
Pre-culture: The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 mL) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C to provide an inoculum in the log phase of growth, characterised by a cell density of 3.28 x 106 cells/mL
Culture conditions: Conical flasks (250 mL) each containing control or test culture (100 mL) were placed in an illuminated orbital incubator according to a random number sequence. The cultures were incubated, without renewal of medium for 72 hours under continuous illumination (nominally 4440 to 8880 lux.) provided by 6 x 30 W “cool white” 1 metre fluorescent tubes. The temperature was maintained at 21 to 24°C. Temperature and pH of control and test flasks at the start and end of the test were recorded. Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at a nominal 150 cycles per minute. The minimum and maximum temperature and light intensity (four corner positions and in a central position of the random block design) within the test area were determined each day. To minimise the impact of differences in light intensity across the test area on algal growth, control and test flasks were re-positioned in the test area each day during the definitive test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23.3 - 25.6°C

pH:

5.28 - 7.13

Nominal and measured concentrations:

Nominal: 0 and 100 mg TOS/L (corresponding to 22.2. mg aep/L)
Measured: At the start of the test, enzyme recovery ranged between 75 and 76% of the nominal value. After 72 hours, the recovery decreased to 11% of nominal.
After 72 hours, analysis of a sample of test medium containing Xylanase, PPQ 30189 which had been incubated without algal cells showed maintenance of enzyme levels (63 to 69%). These results indicate that the presence of algal cells affected the stability of the test substance.

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks (250 mL)
- Type (delete if not applicable): loosely plugged with a foam bung
- Material, size, fill volume: glas, 250 mL, 100 mL
- Initial cells density: 1*10^4 cells/mL
- Control end cells density: 2.4*10^6 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: fitered, dechlorinated tap water, which has been softened and treated by reverse-osmosis before microfiltration and purification (resistivity of 18 Megohm/cm)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes to pH 6.0 ± 0.2 using 0.1 to 1.0 M HCl
- Photoperiod: continuous illumination
- Light intensity and quality: 6492 - 6666 lux (mean valus) provided by 6 x 30 W "cool white" 1 metre fluorescent tubes


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken from control and test flasks at 24, 48 and 72 hours and the cell densities
measured using a Coulter Z Series Particle Count and Size Analyser.
The estimate of cell numbers in each sample was based on the mean of three consecutive counts, corrected for background counts of uninoculated dilution or test media as appropriate; this procedure was employed when counts of the test media on Day 1 of the definitive test indicated a higher particulate count than expected.


TEST CONCENTRATIONS
- Test concentrations: 0 and 100 mg TOS/L or 22.2 mg aep/L

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 22.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 22.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

active enzyme protein

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 22.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 22.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No microscopic abnormalities of the cells were detected, but motile organisms were evident in test media at nominal concentrations of 100 mg TOS/L; these were attributed to the nature of the test substance.
- Any stimulation of growth found in any treatment: no

Reported statistics and error estimates:

The data were compiled in an Excel spreadsheet and analysed using SAS 9.1 (SAS Institute 2002) using nominal concentrations.
The areas under the growth curve were divided by initial counts and total times to give AUCP (Area Under the Curve expressed as a Proportion of the initial cell count), where a value of 1 represents no growth and a value of 0 represents complete toxicity (all algae killed). In order to estimate the concentration at which 50% inhibition of growth occurred (EC50), sigmoidal curves were fitted to AUCP and growth rate. For both variables, 0% inhibition was defined as the control mean and 100% inhibition was defined as no growth. The minimum of the curve (for infinite concentration) was bounded between 0 and 1 for AUCP and between -1000 and 0 for growth rate.

The EC10 and EC50 values could not be calculated because no inhibition of growth was noted.

For AUC and growth rate, the t-test was used to compare the treated group with the control.

Yield was calculated for each test vessel as the final cell density (after 72 hours) minus the presumed initial cell density of 1 x 104 cells/mL. A mean yield value for each test concentration was calculated and the percentage inhibition was determined using the formula below:
% Inhibition = ((YC - YT) / YC) x 100
Where YC = mean value for yield in the control group
YT = value for yield in treatment replicate

For evaluation of effect on area under the curve, growth rate and yield, the t-test was used to compare the treated group with the control.

The mean coefficient of variation (CoV) for daily growth rates in control cultures ranged between 5.03 and 11.6% during the definitive test and the CoV for the average specific growth rates of control culture was 2.24% during the 72 hour exposure period.

Validity criteria fulfilled:

not specified

Conclusions:

Xylanase, batch PPQ 30189 was not inhibitory to algal growth rate of the unicellular green alga Pseudokirchneriella subcapitata. Hence no EC50 value could be determined and the NOEC is the highest concentration tested i.e. 100 mg TOS/L or 22.2 mg aep/L.

Executive summary:

The effect of Xylanase, PPQ 30189 on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed under non-axenic conditions. The active component of the test substance is an enzyme, which was known to be pH sensitive with reduced enzymatic activity under alkaline conditions. In order to maintain the stability of the enzyme the dilution medium used in the study was adjusted to pH 6 before use. The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Directive 761/2009 Part C, Method 3 “Algal Inhibition Test” and Procedure 201 of the “Guidelines for Testing of Chemicals” of the Organisation for Economic Co-operation and Development: Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (2006). Replicate algal cultures, with an initial cell density of 1 x 10⁴/mL, were exposed to Xylanase, PPQ 30189 dispersed in algal nutrient medium at a nominal concentration of 100 mg TOS (Total Organic Solids)/L or 22.2 mg aep (active enzyme protein)/L. The test medium was prepared in OECD medium by the direct addition of the test substance to the dilution medium. The cultures were incubated in an orbital incubator under continuous illumination of approximately 6492 to 6666 lux (mean values) at temperatures ranging from 22.9 to 25.2°C for 72 hours. At the request of the Sponsor, the test concentrations were verified by analysis of the enzyme concentrations, which was performed at the Sponsor’s laboratory. Cell numbers were counted daily to monitor growth.

The test results are expressed in terms of the area under the growth curve, growth rate and yield. The following values were derived from the data:

	Nominal Xylanase, PPQ 30189 concentration (mg aep/L)
Area under the growth curve EbC50 (72 h)	>22.2
Average specific growth rate ErC50 (0 - 72 h)	>22.2
Yield EyC50 (0 - 72 h)	>22.2
“No observed effect concentration”	22.2

Description of key information

Xylanase was not toxic to the green alga Pseudokirchneriella subcapitata at the given conditions and hence no EC50 value could be determined. The NOEC was determined to be >= 100 mg TOS/L or 22.2. mg aep/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

22.2 mg/L